Stable Therapeutic Formulations

ABSTRACT

Compositions of and methods for formulating and delivering biologically active agent formulations having enhanced physical stability, and wherein deterioration from the presence of oxygen and/or water is minimized and/or controlled, to yield a stable formulation. The compositions of and methods for formulating and delivering biologically active agents of the present invention further facilitate their incorporation into a biocompatible coating which can be employed to coat a stratum-corneum piercing microprojection, or a plurality of stratum-corneum piercing microprojections of a delivery device, for delivery of the biocompatible coating through the skin of a subject, thus providing an effective means of delivering the biologically active agents.

RELATED APPLICATIONS

The present application claims priority from U.S. ProvisionalApplication No. 60/754,948 filed on Dec. 28, 2005, the contents of whichare herein incorporated by reference in their entirety.

FIELD OF THE PRESENT INVENTION

The present invention relates generally to biologically active agentcompositions and methods for formulating and delivering suchcompositions. More particularly, the present invention relates tocompositions of and methods for formulating and delivering physicallystabilized biologically active agent compositions by minimizing theexposure of such biologically active agent compositions to oxygen andwater.

BACKGROUND OF THE INVENTION

A great number and variety of biologically active agents are known inthe art to have therapeutic benefits when delivered appropriately to apatient having a condition upon which such biologically active agentscan exert a beneficial effect. These biologically active agents compriseseveral broad classes, including, but not limited to peptides orproteins, such as hormones, proteins, antigens, repressors/activators,enzymes, and immunoglulins, among others. Therapeutic applicationsinclude treatment of cancer, hypercalcemia, Paget's disease,osteoporosis, diabetes, cardiac conditions, including congestive heartfailure, sleep disorders, Chronic Obstructive Pulmonary Disease (COPD)and anabolic conditions, to name a few.

In the art, formulating such biologically active agent formulations in atherapeutically effective and commercially viable manner has beenproblematic, due in part, to the tendency of many biologically activeagents to deteriorate in the presence of oxygen and water. Particularlysusceptible to oxidation include the amino acids methionine andcysteine. Water causes degradation of large number of biological agents.This affects particularly peptides and proteins as a result ofhydrolysis of the amide bond.

References have been published which discuss the effects of oxidationand hydrolysis on biologically active agents during manufacture andstorage. For example, Pikal M J, Dellerman K, Roy M L. Formulation andstability of freeze-dried proteins: effects of moisture and oxygen onthe stability of freeze-dried formulations of human growth hormone. DevBiol Stand. 1992;74:21-38; Lai, Mei C.; Hageman, Michael J.; Schowen,Richard L.; Borchardt, Ronald T.; Topp, Elizabeth M. Chemical Stabilityof Peptides in Polymers. 1. Effect of Water on Peptide Deamidation inPoly(vinyl alcohol) and Poly(vinyl pyrrolidone) Matrixes. Journal ofPharmaceutical Sciences (1999), 88(10), 1073-1080, address howbiological active agents experience deterioration due to oxidation orhydrolysis when exposed to air or water over extended periods of time.

The deterioration of biologically active agents in formulations isparticularly problematic when biologically active agents areadministered by transdermal delivery. The word “transdermal”, as usedherein, is a generic term that refers to delivery of an active agent(e.g., a therapeutic agent, such as a drug, pharmaceutical, peptide,polypeptide or protein) through the skin to the local tissue or systemiccirculatory system without substantial cutting or penetration of theskin, such as cutting with a surgical knife or piercing the skin with ahypodermic needle. Transdermal agent delivery includes delivery viapassive diffusion as well as delivery based upon external energysources, such as electricity (e.g., iontophoresis) and ultrasound (e.g.,phonophoresis).

Numerous transdermal agent delivery systems and apparatus have beendeveloped that employ tiny skin piercing elements to enhance transdermalagent delivery. Examples of such systems and apparatus are disclosed inU.S. Pat. Nos. 5,879,326, 3,814,097, 5,250,023, 3,964,482, Reissue No.25,637, and PCT Publication Nos. WO 96/37155, WO 96/37256, WO 96/17648,WO 97/03718, WO 98/11937, WO 98/00193, WO 97/48440, WO 97/48441, WO97/48442, WO 98/00193, WO 99/64580, WO 98/28037, WO 98/29298, WO98/29365 and US Publication Nos. US2004/0062813, US2004/0265354,US2005/0090009, US2005/0106209, US20050123507, US2005/0226922,US2005/0256045, and US2005/026601 1; all incorporated herein byreference in their entirety.

The disclosed systems and apparatus employ piercing elements of variousshapes and sizes to pierce the outermost layer (i.e., the stratumcorneum) of the skin, and thus enhance the agent flux. The piercingelements generally extend perpendicularly from a thin, flat member, suchas a pad or sheet. The piercing elements are typically extremely small,some having a microprojection length of only about 25-400 microns and amicroprojection thickness of only about 5-50 microns. These tinypiercing/cutting elements make correspondingly smallmicroslits/microcuts in the stratum corneum for enhanced transdermalagent delivery therethrough. The active agent to be delivered isassociated with one or more of the microprojections, usually by coatingthe microprojections with the formulation or by the use of a reservoirthat communicates with the stratum corneum after the microslits areformed.

The current manufacturing and packaging processes, however, areproblematic, especially where the microprojections are coated with theformulation by drying the formulation on the microprojections, asdescribed in U.S. patent application Publication No. 2002/0128599. Theformulation is usually an aqueous formulation. During the dryingprocess, all volatiles, including water are mostly removed, however, thefinal solid coating still contains typically about 3% water. Thepresence of water can lead to deterioration of the biologically activeagent in the formulation because of hydrolysis.

The current manufacturing and packaging processes are also problematicbecause oxygen is present during each phase. While the manufacturingphase is a relatively short period of time, the packaging and storagephase can be quite lengthy. Storage times of transdermal deliverysystems are likely to be for lengthy periods of time before they areused (i.e., extended shelf life of several months is not uncommon). Thebiologically active agents in the coatings, therefore, are subject tooxidation and deterioration. For purposes of this application, referenceto the term “package” or “packaging” will be understood to also includereference to “storage” or “storing”.

Accordingly, physical stabilization, especially minimizing the exposureof the biologically active agent formulations over time to oxidation andhydrolysis, is an important step in assuring efficacy of the therapeuticagents, particularly when the mode of delivery of the therapeutic agentis via a transdermal delivery device having a plurality ofmicroprojections coated with an agent containing biocompatible coating.

It would therefore be desirable to provide compositions of and methodsfor formulating and delivering biologically active agents havingenhanced physical stability.

It would be further desirable to provide compositions of and methods forformulating and delivering biologically active agents whereindeterioration of the biologically active agent from oxygen and/or wateris minimized and/or controlled.

It would be further desirable to provide compositions of and methods forformulating and delivering biologically active agents that exhibitmaximal or optimal shelf lives.

SUMMARY OF THE INVENTION

The present invention provides biologically active agents havingenhanced physical stability, wherein the biologically active agents arecoated on a transdermal delivery device having a plurality ofskin-piercing microprojections that are adapted to deliver the agentthrough the skin of a subject and the device is manufactured and/orpackaged in a dry inert atmosphere and/or a partial vacuum.

In accordance with the compositions of and methods for formulating anddelivering physically stable biologically active agent formulations ofthe present invention, it has been found that the manufacture and/orpackaging of the formulations in a dry inert atmosphere and/or a partialvacuum, substantially free of oxygen and water, substantially reduces oreliminates undesirable deterioration of the biologically active agent.

The present invention provides compositions of and methods forformulating and delivering biologically active agents whereindeterioration from damaging oxygen and/or water is minimized and/orcontrolled.

The present invention also provides compositions of and methods forformulating and delivering biologically active agents that have maximalor optimal shelf lives.

The present invention further provides biologically active agents havingenhanced physical stability, wherein the biologically active agents arecontained in a biocompatible coating that is disposed on a transdermaldelivery device having a plurality of skin-piercing microprojectionsthat are adapted to deliver the agent through the skin of a subject.

The present invention also provides compositions of and methods forformulating and delivering biologically active agent formulationswherein the formulations are stabilized during their manufacture andstorage by the presence of a dry inert atmosphere and/or a partialvacuum, substantially free of oxygen and/or water.

As well, the present invention provides methods for using a dry inertatmosphere and/or a partial vacuum during manufacture and/or packagingto stabilize biologically active agent formulations.

In one embodiment of the invention, there are provided compositions ofand methods for formulating and delivering biologically active agentsthat exhibit improved or optimal physical stability, and which improvedor optimal physical stability enhances shelf life of formulationscontaining the therapeutic agents. The present invention also providesfor compositions of and methods for formulating and deliveringbiologically active agent formulations that have been incorporated in abiocompatible coating that is coated onto a plurality of stratumcorneum-piercing microprojections of a transdermal delivery device, thedelivery device exhibiting improved or optimal physical stability.

In one embodiment of the present invention, the compositions of andmethods for formulating and delivering biologically active agentformulations are suitable for use with a variety of delivery means(e.g., systemic or local delivery), including oral (bolus), oral (timedor pattern release), infusion, injection, subcutaneous implant,pulmonary, mucosal (oral mucosa, ocular, nasal, rectal, vaginal),passive, active and balistic transdermal delivery. Other local delivery,such as treatment of otitis, skin, scalp, nail fungal, bacterial andviral infections, are also within the scope of the invention.

In a preferred embodiment, the compositions of and methods forformulating and delivering biologically active agents are particularlysuitable for transdermal delivery using a microprojection deliverydevice, wherein the biologically active agents are included in abiocompatible coating that is coated on at least one stratum-corneumpiercing microprojection, preferably a plurality of stratum-corneumpiercing microprojections of a microprojection delivery device.

In one embodiment, the compositions of and methods for formulating anddelivering biologically active agents are particularly suitable fortransdermal delivery using a microprojection delivery device, whereinthe biologically active agents are included in a biocompatible coatingthat is manufactured and/or packaged in a dry inert atmosphere,preferably nitrogen or argon.

In one embodiment, the compositions of and methods for formulating anddelivering biologically active agents are particularly suitable fortransdermal delivery using a microprojection delivery device, whereinthe biologically active agents are included in a biocompatible coatingthat is coated on at least one stratum-corneum piercing microprojection,preferably a plurality of stratum-corneum piercing microprojections of amicroprojection delivery device, and manufactured and/or packaged in adry inert atmosphere, preferably nitrogen and argon.

In one embodiment, the compositions of and methods for formulating anddelivering biologically active agents are particularly suitable fortransdermal delivery using a microprojection delivery device, whereinthe biologically active agents are included in a biocompatible coatingthat is manufactured and/or packaged in a dry inert atmosphere,preferably nitrogen or argon, and in the presence of a desiccant oroxygen absorber.

In one embodiment, the compositions of and methods for formulating anddelivering biologically active agents are particularly suitable fortransdermal delivery using a microprojection delivery device, whereinthe biologically active agents are included in a biocompatible coatingthat is manufactured and/or packaged in a foil lined chamber having adry inert atmosphere, preferably nitrogen, and a desiccant or oxygenabsorber.

In one embodiment, the compositions of and methods for formulating anddelivering biologically active agents are particularly suitable fortransdermal delivery using a microprojection delivery device, whereinthe biologically active agents are included in a biocompatible coatingthat is manufactured and/or packaged in a partial vacuum.

In one embodiment, the compositions of and methods for formulating anddelivering biologically active agents are particularly suitable fortransdermal delivery using a microprojection delivery device, whereinthe biologically active agents are included in a biocompatible coatingthat is manufactured and/or packaged in a dry inert atmosphere,preferably nitrogen, and a partial vacuum.

In one embodiment, the compositions of and methods for formulating anddelivering biologically active agents are particularly suitable fortransdermal delivery using a microprojection delivery device, whereinthe biologically active agents are included in a biocompatible coatingthat is manufactured and/or packaged in a foil lined chamber having adry inert atmosphere, preferably nitrogen, a partial vacuum, and adesiccant or oxygen absorber.

In a preferred embodiment, the biologically active agent comprises hPTH.A particularly preferred form is hPTH(1-34) and analogs thereof.

In another preferred embodiment, the resultant formulation of stablebiologically active agents, is incorporated in a biocompatible coatingused to coat at least one stratum-corneum piercing microprojection,preferably a plurality of stratum-corneum piercing microprojections, oran array thereof, or a delivery device. Typically, the coating processis carried out in a series of coating steps, with a drying step betweeneach coating step, as disclosed, for example in U.S. Pat. Pub. No.2002/0132054, to Trautman et al.; the disclosure of which isincorporated by reference herein. The coated microprojections arepackaged in a dry inert atmosphere and/or a partial vacuum.

In accordance with a further embodiment of the invention, an apparatusor device for transdermally delivering the stable biologically activeagents comprises a microprojection member that includes a plurality ofmicroprojections that are adapted to pierce through the stratum corneuminto the underlying epidermis layer, or epidermis and dermis layers, themicroprojection member having a biocompatible coating disposed thereonthat includes a formulation containing the stable biologically activeagents.

In accordance with one embodiment of the invention, a method formanufacturing biologically active agent formulations comprises thefollowing steps: (i) providing a microprojection member having aplurality of microprojections, (ii) providing a formulation ofbiologically active agent; (iii) forming a biocompatible coatingformulation that includes the formulation of biologically active agent,(iv) coating the microprojection member with the biocompatible coatingformulation to form a biocompatible coating; and (v) packaging thebiocompatible coating under dry inert atmospheric conditions and/or apartial vacuum. In a preferred embodiment a desiccant is included in thepackaging.

In accordance with one embodiment of the invention, a method fordelivering biologically active agent formulations comprises thefollowing steps: (i) providing a microprojection member having aplurality of microprojections, (ii) providing a formulation ofbiologically active agent; (iii) forming a biocompatible coatingformulation that includes the formulation of biologically active agent,(iv) coating the microprojection member with the biocompatible coatingformulation to form a biocompatible coating; (v) packaging thebiocompatible coating under dry inert atmospheric conditions and/or apartial vacuum; and (vi) applying the coated microprojection member tothe skin of a subject. In a preferred embodiment a desiccant is includedin the packaging.

BRIEF DESCRIPTION OF THE DRAWINGS

Further features and advantages will become apparent from the followingand more particular description of the preferred embodiments of theinvention, as illustrated in the accompanying drawings, and in whichlike referenced characters generally refer to the same parts or elementsthroughout the views, and in which:

FIG. 1 is a perspective view of a portion of one example of amicroprojection array upon which a biocompatible coating having abiologically active agent formulation can be deposited;

FIG. 2 is a perspective view of the microprojection array shown in FIG.1 with a biocompatible coating deposited onto the microprojections;

FIG. 2A is a cross-sectional view of a single microprojection takenalong line 2A-2A in FIG. 1;

FIG. 3 is a schematic illustration of a skin proximal side of amicroprojection array, illustrating the division of the microprojectionarray into various portions, according to the invention;

FIG. 4 is a side plane view of a skin proximal side of a microprojectionarray, illustrating the division of the microprojection array intovarious portions, according to the invention;

FIG. 5 is a side sectional view of a microprojection array illustratingan alternative embodiment of the invention, wherein differentbiocompatible coatings may be applied to different microprojections;

FIG. 6 is a perspective view of the microprojection array in foilpackaging with a desiccant enclosed.

FIG. 7 shows long-term stability of Macroflux® PTH in differentPackaging Conditions.

FIG. 8 shows % BNP Purity by HPLC following e-beam Treatment (T=0).

FIG. 9 shows BNP Degradation Profile by HPLC at T=0.

FIG. 10 shows a comparison of E-beam treatment degradation profiles fromprevious and Current Studies

FIG. 11 shows 1M stability data following E-beam treatment.

FIG. 12 shows degradation profile following E-beam treatment.

FIG. 13 shows total hBNP % Peak Area Purity (T=0).

FIG. 14 shows HPLC degradation profile (T=0).

FIG. 15 shows total hBNP % Peak Area Purity (T=1M, 25C).

FIG. 16 shows HPLC degradation profile (T=1M, 25C).

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particularlyexemplified materials, formulations, methods or structures as such may,of course, vary. Thus, although a number of materials and methodssimilar or equivalent to those described herein can be used in thepractice of the present invention, the preferred materials and methodsare described herein.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments of the invention only andis not intended to be limiting.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one having ordinaryskill in the art to which the invention pertains.

Further, all publications, patents and patent applications cited herein,whether supra or infra, are hereby incorporated by reference in theirentirety.

Finally, as used in this specification and the appended claims, thesingular forms “a, “an” and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “atherapeutic agent” includes two or more such agents; reference to “amicroprojection” includes two or more such microprojections and thelike.

Definitions

The term “degradation”, as used herein, means the purity of thebiological agent decreases from an initial time point.

The terms “desiccant” and “oxygen absorbers” are used interchangeablyherein. Unless otherwise clear from the context, the noted terms referto a agent that absorbs water, usually a chemical agent.

The term “transdermal”, as used herein, means the delivery of an agentinto and/or through the skin for local or systemic therapy.

The term “deteriorate”, as used herein, means that the biologicallyactive agent is diminished or impaired in quality, character, or value.

The term “minimize”, as used herein, also means reduce.

The term “transdermal flux”, as used herein, means the rate oftransdermal delivery.

The term “stable”, as used herein to refer to an agent formulation,means the agent formulation is not subject to undue chemical or physicalchange, including decomposition, breakdown, or inactivation. “Stable” asused herein to refer to a coating also means mechanically stable, i.e.not subject to undue displacement or loss from the surface upon whichthe coating is deposited.

The terms “therapeutic agent” and “agent”, as used herein, mean andinclude a pharmaceutically active agent and/or a composition of matteror mixture containing an active agent, which is pharmaceuticallyeffective when administered in a therapeutic-effective amount. Aspecific example of a biologically active active agent is HPTH. It is tobe understood that more than one “agent” can be incorporated into thetherapeutic agent formulation(s) of the present invention, and that theterms “agent” and “therapeutic agent” do not exclude the use of two ormore such agents.

The terms “therapeutic-effective” or “therapeutically-effective amount”,as used herein, refer to the amount of the biologically active agentneeded to stimulate or initiate the desired beneficial result. Theamount of the biologically active agent employed in the coatings of theinvention will be that amount necessary to deliver an amount of thebiologically active agent needed to achieve the desired result. Inpractice, this will vary widely depending upon the particularbiologically active agent being delivered, the site of delivery, and thedissolution and release kinetics for delivery of the biologically activeagent into skin tissues.

The term “coating formulation”, as used herein, means and includes afreely flowing composition or mixture, which is employed to coat adelivery surface, including one or more microprojections and/or arraysthereof.

The term “biocompatible coating”, as used herein, means and includes acoating formed from a “coating formulation” that has sufficient adhesioncharacteristics and no (or minimal) adverse interactions with thebiologically active agent.

The term “microprojections”, as used herein, refers to piercing elementsthat are adapted to pierce or cut into and/or through the stratumcorneum into the underlying epidermis layer, or epidermis and dermislayers, of the skin of a living animal, particularly a mammal and, moreparticularly, a human.

The term “microprojection member”, as used herein, generally connotes amicroprojection array comprising a plurality of microprojectionsarranged in an array for piercing the stratum corneum. Themicroprojection member can be formed by etching or punching a pluralityof microprojections from a thin sheet and folding or bending themicroprojections out of the plane of the sheet to form a configuration.The microprojection member can also be formed in other known manners,such as by forming one or more strips having microprojections along anedge of each of the strip(s), as disclosed in U.S. Pat. No. 6,050,988,which is hereby incorporated by reference in its entirety.

Microprojection members that can be employed with the present inventioninclude, but are not limited to, the members disclosed in U.S. Pat. Nos.6,083,196, 6,050,988 and 6,091,975, and U.S. Patent Application Pub. No.2002/0016562, which are incorporated by reference herein in theirentirety. As will be appreciated by one having ordinary skill in theart, where a microprojection array is employed, the dose of thetherapeutic agent that is delivered can also be varied or manipulated byaltering the microprojection array (or patch) size, density, etc.

As discussed above in the Background section, current manufacturing andpackaging processes for delivery devices involving microprojections areproblematic, especially where the microprojections are coated with aformulation by drying the formulation on the microprojections, asdescribed in U.S. patent application Publication No. 2002/0128599. Theformulation is usually an aqueous formulation. During the dryingprocess, all volatiles, including water are mostly removed, however, thefinal solid coating still contains typically about 3% water. Thepresence of water can lead to deterioration of the biologically activeagent in the formulation because of hydrolysis.

The current manufacturing and packaging processes are also problematicbecause oxygen is present during each phase. While the manufacturingphase is a relatively short period of time, the packaging and storagephase can be quite lengthy. Storage times of transdermal deliverysystems are likely to be for lengthy periods of time before they areused (i.e., extended shelf life of several months is not uncommon). Thebiologically active agents in the coatings, therefore, are subject tooxidation and deterioration.

For purposes of this application, reference to the term “package” or“packaging” will be understood to also include reference to “storage” or“storing”.

Accordingly, physical stabilization, especially minimizing the exposureof the biologically active agent formulations over time to oxidation andhydrolysis, is an important step in assuring efficacy of the therapeuticagents, particularly when the mode of delivery of the therapeutic agentis via a transdermal delivery device having a plurality ofmicroprojections coated with an agent containing biocompatible coating.

The above noted publication, however, does not disclose a formulationof, or technique for, physically stabilizing formulations ofoxygen-sensitive biologically active agents, in particular, mitigatingor eliminating the presence of oxygen during the packaging oftransdermal delivery devices, and resultant unwanted oxidation of thebiologically active agents. In particular, the noted publication doesnot disclose a formulation of, or technique for, physically stabilizingbiologically active agents by manufacturing and/or packaging thebiologically active agent-containing transdermal delivery devices in adry inert atmosphere (essentially zero water content), which impart tothe formulation stability against undesired changes over time due tooxidation.

Nor does the above noted publication disclose a formulation of, ortechnique for, physically stabilizing formulations of water-sensitivebiologically active agents, in particular, mitigating or eliminating thepresence of water during the packaging of transdermal delivery devices,and resultant unwanted hydrolysis of the biologically active agents. Inparticular, the noted publication does not disclose a formulation of, ortechnique for, physically stabilizing biologically active agents bymanufacturing and/or packaging the biologically active agent-containingtransdermal delivery devices in a partial vacuum, which impart to theformulation stability against undesired changes over time due tohydrolysis.

Improved physical stability of such therapeutic formulations ofbiologically active agents provides not only the benefit of an increasedstorage or shelf life for the therapeutic agent itself, but enhancesefficacy in that once stabilized in accordance with the compositions ofand methods for formulating and delivering of the present invention, thetherapeutic agents become useful in a greater range of possibleformulations, and with a greater variety of therapeutic agent deliverymeans.

As indicated above, the present invention comprises compositions of andmethods for formulating and delivering biologically active agents havingenhanced physical stability, and wherein deterioration from the presenceof oxygen and/or water is minimized and/or controlled. The compositionsof and methods for formulating and delivering biologically active agentformulations further allow for the minimization and/or control ofimpurities and oxidative byproducts to yield a consistent andpredictable composition. The compositions of and methods for formulatingand delivering biologically active agent formulations of the presentinvention further facilitate their incorporation into a biocompatiblecoating which can be employed to coat a stratum-corneum piercingmicroprojection, or a plurality of stratum-corneum piercingmicroprojections of a delivery device, for delivery of the biocompatiblecoating through the skin of a subject, thus providing an effective meansof delivering the biologically active agents.

The above noted publication, nor any other known reference, however,disclose a formulation of or technique for, physically stabilizingformulations of oxygen-sensitive biologically active agents, inparticular, mitigating or eliminating the presence of oxygen during thepackaging of transdermal delivery devices, and resultant unwantedoxidation of the biologically active agents. In particular, the notedpublication, nor any other known reference disclose a formulation of, ortechnique for, physically stabilizing biologically active agents bymanufacturing and/or packaging the biologically active agent-containingtransdermal delivery devices in a dry inert atmosphere (essentially zerowater content), which impart to the formulation stability againstundesired changes over time due to oxidation.

Nor does the above noted publication, nor any other known referencedisclose a formulation of, or technique for, physically stabilizingformulations of water-sensitive biologically active agents, inparticular, mitigating or eliminating the presence of water during thepackaging of transdermal delivery devices, and resultant unwantedhydrolysis of the biologically active agents. In particular, the notedpublication, nor any other known reference disclose a formulation of, ortechnique for, physically stabilizing biologically active agents bymanufacturing and/or packaging the biologically active agent-containingtransdermal delivery devices in a partial vacuum, which impart to theformulation stability against undesired changes over time due tohydrolysis.

Improved physical stability of such therapeutic formulations ofbiologically active agents provides not only the benefit of an increasedstorage or shelf life for the therapeutic agent itself, but enhancesefficacy in that once stabilized in accordance with the compositions ofand methods for formulating and delivering of the present invention, thetherapeutic agents become useful in a greater range of possibleformulations, and with a greater variety of therapeutic agent deliverymeans.

According to one embodiment, the present invention comprises abiologically active agent formulation wherein the deterioration byoxygen and/or water is minimized and/or controlled by the manufactureand/or packaging of the biologically active agent formulation in a dryinert atmosphere. Preferably the biologically active agent is containedin a dry inert atmosphere in the presence of a desiccant. Morepreferably, the biologically active agent is contained in a dry inertatmosphere in the presence of a desiccant in a foil-lined chamber.

According to one embodiment, the present invention comprises abiologically active agent formulation wherein the deterioration byoxygen and/or water is minimized and/or controlled by the manufactureand/or packaging of the biologically active agent formulation in apartial vacuum. Preferably the biologically active agent is contained ina partial vacuum in the presence of a desiccant. More preferably, thebiologically active agent is contained in a partial vacuum in thepresence of a desiccant in a foil-lined chamber.

According to one embodiment, the present invention comprises abiologically active agent formulation wherein the deterioration byoxygen and/or water is minimized and/or controlled by the manufactureand/or packaging of the biologically active agent formulation in a dryinert atmosphere and a partial vacuum. Preferably the biologicallyactive agent is contained in a dry inert atmosphere and a partial vacuumin the presence of a desiccant. More preferably, the biologically activeagent is contained in a dry inert atmosphere and a partial vacuum in thepresence of a desiccant in a foil-lined chamber.

Generally, in the noted embodiments of the present invention, the dryinert atmosphere is nitrogen, but can also be any other inert atmosphereknown to those of skill in the art such as argon, helium, neon, krypton,carbon dioxide. For improved stability of the product, the inertatmosphere should be essentially zero water content. For example,nitrogen gas of essentially zero water content (dry nitrogen gas) can beprepared very simply by electrically controlled boiling of liquidnitrogen. Purge systems can be also used to reduce moisture or oxygencontent.

Packaging under a partial vacuum is known to those skilled in the art. Apreferred range for a partial vacuum is from about 0.01 to about 0.3atmospheres.

As discussed above, the biologically active agent formulations aregenerally prepared as a solid coating by drying a formulation on themicroprojection, as described in U.S. patent application Publication No.2002/0128599. The formulation is usually an aqueous formulation. Duringa drying process, all volatiles, including water are mostly removed,however, the final solid coating still contains typically about 3%water.

The present invention reduces the oxygen and/or water content present inthe formulations. Oxygen and/or water content are reduced by the use ofa dry inert atmosphere and/or a partial vacuum. In a solid coating on amicroprojection array, the drug is typically present in an amount ofless than about 1 mg per unit dose. With the addition of excipients, thetotal mass of solid coating is less than 3 mg per unit dose. The arrayis usually present on an adhesive backing, which is attached to adisposable polymeric retainer ring. This assembly is packagedindividually in a pouch or a polymeric housing. In addition to theassembly, this package contains a dead volume that represents a volumeof at least 3 mL. This large volume (as compared to that of the coating)acts as a partial sink for water. For example, at 20 degree C., theamount of water present in a 3 mL atmosphere as a result of its vaporpressure would be about 0.05 mg at saturation, which is typically theamount of residual water that is present in the solid coating afterdrying. Therefore, storage in a dry inert atmosphere and/or a partialvacuum will further reduce the water content of the coating resulting inimproved stability.

In addition, desiccants and oxygen absorbers can be incorporated in thepackaging to further reduce oxygen and water content. The desiccant oroxygen absorber can be any known to those skilled in the art. Somecommon desiccants or oxygen absorbers include, but are not limited tocalcium oxide, clay desiccant, calcium sulfate, and silica gel. Thedesiccant or oxygen absorber is preferably one that can be placed withthe biologically active agent-containing formulation in the presence ofan inert atmosphere in a foil-lined chamber.

The biologically active agent formulation is preferably packaged in afoil-lined chamber after the biologically active agent formulation isprepared, and preferably after the biologically active agent formulationis coated onto the microprojection array delivery device. In oneembodiment, the coated delivery device is placed in a foil-lined chamberas depicted in FIG. 6 and discussed below in the Example section in moredetail. In this embodiment, a desiccant or oxygen absorber is attachedto a foil lid and the chamber is purged with dry nitrogen prior to thefoil delivery device-containing foil chamber being sealed by the foillid.

Therapeutic Agents

A great number and variety of biologically active agents are known inthe art to have therapeutic benefits when delivered appropriately to apatient having a condition upon which such therapeutic agents can exerta beneficial effect.

Suitable biologically active agents include therapeutic agents in allthe major therapeutic areas including, but not limited to:anti-infectives, such as antibiotics and antiviral agents; analgesics,including fentanyl, sufentanil, remifentanil, buprenorphine andanalgesic combinations; anesthetics; anorexics; antiarthritics;antiasthmatic agents, such as terbutaline; anticonvulsants;antidepressants; antidiabetic agents; antidiarrheals; antihistamines;anti-inflammatory agents; antimigraine preparations; antimotion sicknesspreparations such as scopolamine and ondansetron; antinauseants;antineoplastics ; antiparkinsonism drugs; antipruritics; antipsychotics;antipyretics; antispasmodics, including gastrointestinal and urinary;anticholinergics; sympathomimetrics; xanthine derivatives;cardiovascular preparations, including calcium channel blockers such asnifedipine; beta blockers; beta-agonists such as dobutamine andritodrine; antiarrythmics; antihypertensives, such as atenolol; ACEinhibitors, such as ranitidine; diuretics; vasodilators, includinggeneral, coronary, peripheral, and cerebral; central nervous systemstimulants; cough and cold preparations; decongestants; diagnostics;hormones, such as parathyroid hormone; hypnotics; immunosuppressants;muscle relaxants; parasympatholytics; parasympathomimetrics;prostaglandins; proteins; peptides; psychostimulants; sedatives; andtranquilizers. Other suitable agents include vasoconstrictors,anti-healing agents and pathway patency modulators.

Further specific examples of agents include, without limitation, growthhormone release hormone (GHRH), growth hormone release factor (GHRF),insulin, insultropin, calcitonin, octreotide, endorphin, TRN, NT-36(chemical name: N-[[(s)-4-oxo-2-azetidinyl]carbonyl]-L-histidyl-L-prolinamide), liprecin, pituitary hormones (e.g.,HGH, HMG, desmopressin acetate, etc), follicle luteoids, aANF, growthfactors such as growth factor releasing factor (GFRF), bMSH, GH,somatostatin, bradykinin, somatotropin, platelet-derived growth factorreleasing factor, asparaginase, bleomycin sulfate, chymopapain,cholecystokinin, chorionic gonadotropin, erythropoietin, epoprostenol(platelet aggregation inhibitor), glucagon, HCG, hirulog, hyaluronidase,interferon alpha, interferon beta, interferon gamma, interleukins,interleukin-10 (IL-10), erythropoietin (EPO), granulocyte macrophagecolony stimulating factor (GM-CSF), granulocyte colony stimulatingfactor (G-CSF), glucagon, leutinizing hormone releasing hormone (LHRH),LHRH analogs (such as goserelin, leuprolide, buserelin, triptorelin,gonadorelin, and napfarelin, menotropins (urofollitropin (FSH) and LH)),oxytocin, streptokinase, tissue plasminogen activator, urokinase,vasopressin, deamino [Val4, D-Arg8] arginine vasopressin, desmopressin,corticotropin (ACTH), ACTH analogs such as ACTH (1-24), ANP, ANPclearance inhibitors, angiotensin II antagonists, antidiuretic hormoneagonists, bradykinn antagonists, ceredase, CSI's, calcitonin generelated peptide (CGRP), enkephalins, FAB fragments, IgE peptidesuppressors, IGF-1, neurotrophic factors, colony stimulating factors,parathyroid hormone and agonists, parathyroid hormone antagonists,parathyroid hormone (PTH), PTH analogs such as PTH (1-34), prostaglandinantagonists, pentigetide, protein C, protein S, renin inhibitors,thymosin alpha-1, thrombolytics, TNF, vasopressin antagonists analogs,alpha-1 antitrypsin (recombinant), and TGF-beta.

The biologically active agent can also comprise a vaccine, includingviruses and bacteria, protein-based vaccines, polysaccharide-basedvaccine, nucleic acid-based vaccines, and other antigenic agents.Suitable antigenic agents include, without limitation, antigens in theform of proteins, polysaccharide conjugates, oligosaccharides, andlipoproteins. These subunit vaccines in include Bordetella pertussis(recombinant PT accince —acellular), Clostridium tetani (purified,recombinant), Corynebacterium diptheriae (purified, recombinant),Cytomegalovirus (glycoprotein subunit), Group A streptococcus(glycoprotein subunit, glycoconjugate Group A polysaccharide withtetanus toxoid, M protein/peptides linke to toxing subunit carriers, Mprotein, multivalent type-specific epitopes, cysteine protease, C5apeptidase), Hepatitis B virus (recombinant Pre SI, Pre-S2, S,recombinant core protein), Hepatitis C virus (recombinant—expressedsurface proteins and epitopes), Human papillomavirus (Capsid protein,TA-GN recombinant protein L2 and E7 [from HPV-6], MEDI-501 recombinantVLP L1 from HPV-11, Quadrivalent recombinant BLP L1 [from HPV-6],HPV-11, HPV-16, and HPV-18, LAMP-E7 [from HPV-16]), Legionellapneumophila (purified bacterial survace protein), Neisseria meningitides(glycoconjugate with tetanus toxoid), Pseudomonas aeruginosa (syntheticpeptides), Rubella virus (synthetic peptide), Streptococcus pneumoniae(glyconconjugate [1, 4, 5, 6B, 9N, 14, 18C, 19V, 23F] conjugated tomeningococcal B OMP, glycoconjugate [4, 6B, 9V, 14, 18C, 19F, 23F]conjugated to CRM 197, glycoconjute [1, 4, 5, 6B, 9V, 14, 18C, 19F, 23F]conjugated to CRM1970, Treponema pallidum (surface lipoproteins),Varicella zoster virus (subunit, glycoproteins), and Vibrio cholerae(conjugate lipopolysaccharide).

Whole virus or bacteria include, without limitation, weakened or killedviruses, such as cytomegalo virus, hepatitis B virus, hepatitis C virus,human papillomavirus, rubella virus, and varicella zoster, weakened orkilled bacteria, such as bordetella pertussis, clostridium tetani,corynebacterium diptheriae, group A streptococcus, legionellapneumophila, neisseria meningitdis, pseudomonas aeruginosa,streptococcus pneumoniae, treponema pallidum, and vibrio cholerae, andmixtures thereof.

Additional commercially available vaccines, which contain antigenicagents, include, without limitation, flu vaccines, lyme disease vaccine,rabies vaccine, measles vaccine, mumps vaccine, chicken pox vaccine,small pox vaccine, hepatitus vaccine, pertussis vaccine, and diptheriavaccine.

Vaccines comprising nucleic acids include, without limitation,single-stranded and double-stranded nucleic acids, such as, for example,supercoiled plasmid DNA; linear plasmid DNA; cosmids; bacterialartificial chromosomes (BACs); yeast artificial chromosomes (YACs);mammalian artificial chromosomes; and RNA molecules, such as, forexample, mRNA. The size of the nucleic acid can be up to thousands ofkilobases. In addition, in certain embodiments of the invention, thenucleic acid can be coupled with a proteinaceous agent or can includeone or more chemical modifications, such as, for example,phosphorothioate moieties. The encoding sequence of the nucleic acidcomprises the sequence of the antigen against which the immune responseis desired. In addition, in the case of DNA, promoter andpolyadenylation sequences are also incorporated in the vaccineconstruct. The antigen that can be encoded include all antigeniccomponents of infectious diseases, pathogens, as well as cancerantigens. The nucleic acids thus find application, for example, in thefields of infectious diseases, cancers, allergies, autoimmune, andinflammatory diseases.

Suitable immune response augmenting adjuvants which, together with thevaccine antigen, can comprise the vaccine include aluminum phosphategel; aluminum hydroxide; algal glucan: b-glucan; cholera toxin Bsubunit; CRL1005: ABA block polymer with mean values of x=8 and y=205;gamma inulin: linear (unbranched) β-D(2→1)polyfructofuranoxyl-a-D-glucose; Gerbu adjuvant: N-acetylglucosamine-(b1-4)-N-acetylmuramyl-L-alanyl-D-glutamine (GMDP), dimethyldioctadecylammonium chloride (DDA), zinc L-proline salt complex(Zn-Pro-8); Imiquimod(1-(2-methypropyl)-1H-imidazo[4,5-c]quinolin-4-amine; ImmTherÔ:N-acetylglucoaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-glyceroldipalmitate; MTP-PE liposomes: C59H108N6019PNa-3H2O (MTP); Murametide:Nac-Mur-L-Ala-D-Gln-OCH3; Pleuran: b-glucan; QS-21; S-28463: 4-amino-a,a-dimethyl-1H-imidazo[4,5-c]quinoline-1-ethanol; sclavo peptide:VQGEESNDK HCl (IL-1b 163-171 peptide); and threonyl-MDP (TermurtideÔ):N-acetyl muramyl-L-threonyl-D-isoglutamine, and interleukine 18, IL-2IL-12, IL-15, Adjuvants also include DNA oligonucleotides, such as, forexample, CpG containing oligonucleotides. In addition, nucleic acidsequences encoding for immuno-regulatory lymphokines such as IL-18, IL-2IL-12, IL-15, IL-4, IL 10, gamma interferon, and NF kappa B regulatorysignaling proteins can be used.

In another embodiment, suitable counterions are added to the formulationto further improve the stability of the formulation. Examples ofcounterions suitable for formulation with net positively chargedbiologically active agent include, but are not limited to, acetate,propionate, butyrate, pentanoate, hexanoate, heptanoate, levulinate,chloride, bromide, citrate, succinate, maleate, glycolate, gluconate,glucuronate, 3-hydroxyisobutyrate, 2-hydroxyisobutyrate, lactate,malate, pyruvate, fumarate, tartarate, tartronate, nitrate, phosphate,benzene sulfonate, methane sulfonate, sulfate, and sulfonate.Preferably, the counterion mixture is added to the biologically activeagent formulation in an amount sufficient to neutralize the net chargeof the biologically active agent. However, an excess of counterionmixture (either as the acid or the conjugate acid-base) can be added tothe biologically active agent.

In another embodiment of the present invention, the biologically activeagent possesses a net negative charge, and counterion mixture preferablypossesses a net positive charge at the solution pH. Examples ofnegatively-charged biologically active agents include insulin in the pHrange 6-14, VEGF in the pH range 6-14, and insulinotropin in the pHrange 6-14.

In the above embodiment, examples of counterions suitable forformulation with net negatively charged biologically active agentsinclude, but are not limited to, sodiurn, potassium, calcium, magnesium,ammonium, monoethanolamine, diethanolamine, triethanolamine,tromethamine, lysine, histidine, arginine, morpholine, methylglucamine,and glucosanine. The counterion or counterion mixture is preferablyadded to the biologically active agent formulation in an amountsufficient to neutralize the net charge of the biologically activeagent. However, an excess of counterion or counterion mixture (either asthe base or the conjugate acid-base) can be added to the biologicallyactive agent.

In a particularly preferred embodiment of the present invention, thebiologically active agent comprises hPTH. It is particularly preferredto stabilize the hPTH formulation in an inert atmosphere, preferablynitrogen.

In a preferred embodiment, the biologically active agent and counterion(or counterion mixture) is formulated as a solution or suspension in anappropriate solvent. Suitable solvents include water, DMSO, ethanol,isopropanol, DMF, acetonitrile, N-methyl-2-pyrollidone, and mixturesthereof. In addition, the biologically active agent can be in solutionor suspension in a polymeric vehicle, such as EVA or PLGA. As is knownin the art, additional stabilizing additives, such as sucrose andtrehalose, may be present in the formulation.

Various other additives that aid in the delivery, stability or efficacyof the biologically active agents of the present invention can also beadded to the formulations of the invention. Thus, the compositions andformulations of the present invention can contain suitable adjuvants,excipients, solvents, salts, surfactants, buffering agents and othercomponents. Examples of such additives can be found in U.S. patentapplication Nos. 10/880,702 and 10/970,890, the disclosures of which areincorporated by reference herein.

In additional embodiments of the present invention, the biologicallyactive agents, which have been stabilized by minimizing or eliminatingexposure to oxygen after the agents are formulated as a solution orsuspension, and then can be dried, freeze-dried (or lyophilized), spraydried or spray-freeze dried to stabilize for storage.

In another preferred embodiment of the present invention, thebiologically active agent formulations, which have been stabilized byminimizing or eliminating exposure to oxygen and water, are included inbiocompatible coating formulations used to coat a stratum-corneumpiercing microprojection, or plurality of a stratum-corneum piercingmicroprojections, or an array thereof, or delivery device, for deliveryof the biologically active agent through the skin of a patient.Compositions of and methods for formulating biocompatible coatings aredescribed in U.S. Patent Application Pub. No. 2002/0177839 to Cormier etal; U.S. Patent Application Pub. No. 2004/0062813 to Cormier et al andU.S. Patent Application Pub. No. 2002/0132054 to Trautman et al, thedisclosures of which are incorporated herein by reference.

For biologically active agent formulations, particularly thosetherapeutic agents which comprise or include relatively high molecularweight polypeptides or proteins, it is preferred to formulate thebiocompatible coating containing the therapeutic agent, such that awater-soluble, biocompatible polymer, is attached to, or associatedwith, the polypeptide or protein. A particularly preferred method is toform a conjugate of the polymer with the polypeptide or protein. Theattachment of a polymer, such as PEG, to proteins and polypeptidestypically results in improved solubility, improved physical and chemicalstability, lower aggregation tendency and enhanced flow characteristics.Compositions of and methods for formulating biocompatible coatingshaving polymer conjugates of protein and polybiologically active agentsare disclosed in U.S patent application Ser. No. 10/972,231, thedisclosures of which is incorporated herein by reference.

Other compositions of and methods for formulating and deliveringprotein-based therapeutic agent formulations are disclosed in U.S.Patent Application No. 60/585,276, filed July 1, 2004, the disclosure ofwhich is incorporated by reference herein. The noted applicationdiscloses compositions of and methods for formulating hormonetherapeutic agents having a desired pharmacokinetic delivery profile, aswell as the formulation of biocompatible coatings therewith.

In accordance with one embodiment of the invention, a method fordelivering stable biologically active agent formulations comprises thefollowing steps: (i) providing a microprojection member having aplurality of microprojections, (ii) providing a stabilized formulationof biologically active agent; (iii) forming a biocompatible coatingformulation that includes the formulation of stabilized biologicallyactive agent, (iv) coating the microprojection member with thebiocompatible coating formulation to form a biocompatible coating; (v)stabilizing the biocompatible coating by drying; and (vi) applying thecoated microprojection member to the skin of a subject.

FIG. 1 illustrates one embodiment of a stratum corneum-piercingmicroprojection array for use with the compositions and methods forformulating and delivering of the present invention. As shown in FIG. 1,the microprojection array 5 includes a plurality of microprojections 10.The microprojections 10 extend at substantially a 90 degree angle from asheet 12 having openings 14. As shown in FIG. 5, the sheet 12 can beincorporated in a delivery patch including a backing 15 for the sheet12. The backing 15 can further include an adhesive 16 for adhering thebacking 15 and microprojection array 5 to a patient's skin. In thisembodiment, the microprojections 10 are formed by either etching orpunching a plurality of microprojections 10 out of a plane of the sheet12.

The microprojection array 5 can be manufactured of metals, such asstainless steel, titanium, nickel titanium alloys, or similarbiocompatible materials, such as plastics. In a preferred embodiment,the microprojection array is constructed of titanium. Metalmicroprojection members are disclosed in Trautman et al., U.S. Pat. No.6,038,196; Zuck U.S. Pat. No. 6,050,988; and Daddona et al., U.S. Pat.No. 6,091,975, the disclosures of which are herein incorporated byreference.

Other microprojection members that can be used with the presentinvention are formed by etching silicon, by utilizing chip etchingtechniques or by molding plastic using etched micro-molds. Silicon andplastic microprojection members are disclosed in Godshall et al., U.S.Pat. No. 5,879,326, the disclosure of which is incorporated herein byreference.

With such microprojection devices, it is important that thebiocompatible coating having the biologically active agent is applied tothe microprojections homogeneously and evenly, preferably limited to themicroprojections themselves. This enables dissolution of thebiologically active agent in the interstitial fluid once the device hasbeen applied to the skin and the stratum corneum pierced. Additionally,a homogeneous coating provides for greater mechanical stability bothduring storage and during insertion into the skin. Weak and/ordiscontinuous coatings are more likely to flake off during manufactureand storage, and to be wiped of the skin during application.

Additionally, optimal stability and shelf life of the agent is attainedby a biocompatible coating that is solid and substantially dry. However,the kinetics of the coating dissolution and agent release can varyappreciably depending upon a number of factors. It will be readilyappreciated that in addition to being storage stable, the biocompatiblecoating should permit desired release of the therapeutic agent.

Depending on the release kinetics profile, it may be necessary tomaintain the coated microprojections in piercing relation with the skinfor extended periods of time (e.g., up to about 8 hours). This can beaccomplished by anchoring the microprojection member to the skin usingadhesives or by using anchored microprojections, such as described inU.S. Pat. No. 6,230,051, to Cormier et al, the disclosure of which isincorporated by reference herein in its entirety.

Compositions of and methods for formulating biocompatible coatings aredescribed, for example, in U.S. Patent Application Pub. Nos.2002/0128599, 2002/0177839 and 2004/0115167, the disclosures of whichare incorporated herein by reference.

In one embodiment of the present invention, a dip-coating process isemployed to coat the microprojections by partially or totally immersingthe microprojections into the biocompatible coating solution containingthe stable biologically active agent formulation. Alternatively, theentire device can be immersed into the biocompatible coating solution.

In many instances, the stable therapeutic agent within the coating canbe very expensive. Therefore, it may be preferable to only coat the tipsof the microprojections. Microprojection tip coating apparatus andmethods are disclosed in Trautman et al., US Patent Application Pub. No.2002/0132054. The noted publication discloses a roller coating mechanismthat limits the coating to the tips of the microprojection.

As described in the Trautman et al publication, the coating device onlyapplies the coating to the microprojections and not upon thesubstrate/sheet that the microprojections extend from. This may bedesirable in the case where the cost of the active (or beneficial) agentis relatively high and therefore the coating containing the beneficialagent should only be disposed onto parts of the microprojection arraythat will pierce beneath the patient's stratum corneum layer.

The noted coating technique has the added advantage of naturally forminga smooth coating that is not easily dislodged from the microprojectionsduring skin piercing. The smooth cross section of the microprojectiontip coating is more clearly shown in FIG. 2A.

Other coating techniques, such as microfluidic spray or printingtechniques, can also be used to precisely deposit a coating 18 on thetips of the microprojections 10, as shown in FIG. 2.

Other coating methods that can be employed in the practice of thepresent invention include spraying the coating solution onto themicroprojections. Spraying can encompass formation of an aerosolsuspension of the coating composition. In one embodiment, an aerosolsuspension forming a droplet size of about 10 to about 200 picoliters issprayed onto the microprojections and then dried.

The microprojections 10 can further include means adapted to receiveand/or increase the volume of the coating 18 such as apertures (notshown), grooves (not shown), surface irregularities (not shown), orsimilar modifications, wherein the means provides increased surface areaupon which a greater amount of coating may be deposited.

Referring now to FIGS. 3 and 4, there is shown an alternative embodimentof a microprojection array 5. As shown in FIG. 3, the microprojectionarray 5 may be divided into portions illustrated at 60-63, wherein adifferent coating is applied to each portion, thereby allowing a singlemicroprojection array to be utilized to deliver more than one beneficialagent during use.

Referring now to FIG. 4, there is shown a cross-sectional view of themicroprojection array 5, wherein a “pattern coating” has been applied tothe microprojection array 5. As shown, each of the microprojections 10can be coated with a different biocompatible coating and/or a differenttherapeutic agent, as indicated by reference numerals 61-64. That is,separate coatings are applied to the individual microprojections 10. Thepattern coating can be applied using a dispensing system for positioningthe deposited liquid onto the surface of the microprojection array.

The quantity of the deposited liquid is preferably in the range of 0.1to 20 nanoliters/microprojection. Examples of suitable precision-meteredliquid dispensers are disclosed in U.S. Pat. Nos. 5,916,524, 5,743,960,5,741,554 and 5,738,728, the disclosures of which are incorporatedherein by reference.

Microprojection coating solutions can also be applied using ink jettechnology using known solenoid valve dispensers, optional fluid motivemeans and positioning means which are generally controlled by use of anelectric field. Other liquid dispensing technology from the printingindustry or similar liquid dispensing technology known in the art can beused for applying the pattern coating of this invention.

In yet another preferred embodiment, the process of applying abiocompatible coating containing a biologically active agent of theinvention to at least one stratum-corneum piercing microprojection of amicroprojection member, more preferably, to a plurality of suchstratum-corneum piercing microprojections, includes the step of furtherstabilizing the biocompatible coating by drying. The drying step canoccur at ambient (room) temperatures and conditions, or can employtemperatures in the range of 4 to 50° C.

Suitable drying methods and apparatus are disclosed in U.S. PatentApplication No. 60/572,861, filed May 19, 2004, the disclosure of whichis incorporated herein by reference.

According to the invention, a multitude of biologically active activeagents can be subjected to the formulation process and methods of theinvention to provide highly stable biologically active formulations. Ina preferred embodiment of the invention, the therapeutic agent compriseshPTH or an analog thereof.

EXAMPLES

The following studies and examples illustrate the formulations, methodsand processes of the invention. The examples are for illustrativepurposes only and are not meant to limit the scope of the invention inany way.

Example 1

This study demonstrates how a dry inert environment allows formaintaining stability of hPTH(1-34). A delivery device having stratumcorneum piercing microprojections coated with a formulation ofhPTH(1-34) was prepared. The primary packaging for all dosages of thesystems was a heat sealed foil pouch purged with nitrogen gas.

The storage environment within the sealed foil pouches, was assessed byperforming head-space gas analysis, whereby a head-space gas sample isobtained and subjected to quadruple mass spectroscopy analysis for theidentification and quantitation of low molecular weight volatilecompounds, including moisture, oxygen, nitrogen, and argon.

In addition, three foil pouches packaged with fully assembled systems(without desiccant) were assessed in the same manner. Results indicatethat the level of moisture inside the pouch is quite variable from pouchto pouch, 26% to 45% RH(at 22° C. in Table 1), which corresponds to thewater content of 3.5% and 7%, respectively. Notably, all samples reached100% RH if stored at 5° C. Such humidity is considered high and may bedetrimental to hPTH stability. Therefore, reducing head-space humidityis necessary.

In one process, the retainer rings were pre-dried (rings dried in vacuoat 60° C. for at least 48 h prior assembly) or packaged without aretainer ring or adhesive (Table 2). The moisture and oxygen levels weresubstantially reduced—2.2% RH in the sample packaged with a pre-driedretainer ring. In the system without the adhesive or ring, the moisturelevel was also reduced to 9% RH. Therefore, the results indicate thatthe retainer ring is a source of moisture and oxygen; the ring containsa substantial amount of adsorbed water and oxygen that desorbs into thehead-space gas once the pouch has been purged and sealed. Thisillustrates that pre-drying the ring effectively removes the desorbedoxygen and moisture from the ring prior to pouching.

In a different process, desiccant was added directly to the pouch, usingthe 4 Å molecular sieve desiccant of two different sizes andconfigurations: 1) 3.5 g Minipax molecular sieves packaged in Tyveksachets, and 2) 125 mg Desimax adhesive labels affixed to the insidewalls of the foil pouch. The head-space results (Table 3) indicate thatthe 3.5 g 4 Å molecular sieves are effective in reducing pouch humiditywhile the smaller desiccant labels (0.25 g) perform poorly. Desiccantswere manually inserted and handled in ambient air prior to pouching andthe smaller desiccant labels may have reached near moisture saturationeven before packaging.

Since the retainer ring is a source of moisture and a sufficient amountof desiccant is needed, the internal vapor level was assessed of 12Macroflux® systems in the current packaging condition, i.e., coatedarray, Tyvek pouch, 3 Å, 3.5 g desiccant sachet, foil pouch and N2purge. As summarized in Table 4, % RH is extremely low, <1%, in all 12samples, indicating the desiccant is functioning consistently tomaintain the internal head space very dry although the retainer ring canrelease variable amounts of moisture during storage.

Macroflux® PTH systems were sealed in foil pouches in one of the threepackaging configurations: 1) the coated array only with N2 purge; 2) thefully assembled system (coated array+adhesive+retainer ring) with N2purge; 3) the fully assembled system + a 3.5 g desiccant sachet with N2purge. All systems were stored at 25° C. for 12 months. Samples wereremoved at 3, 6, 9, and 12 months for reverse RHPLC analysis.

FIG. 7 summarizes % PTH purity change with time. The fully packagedsystems containing desiccant performed the best. Systems containing onlythe coated array performed well and quite comparable to the fullypackaged systems containing desiccant. According to the head-spaceanalysis mentioned above, the environmental % RH in the system withdesiccant is <1% while that in the system without desiccant can be ashigh as 50%. The coated-array-only system also offers a dry environment,<10% RH (Table 2), as it lacks the retainer ring which is the mainsource of moisture.

Water (or moisture) is known to be able to adversely affectpeptide/protein stability in the solid-state formulation. The humidenvironment enhances moisture adsorption by the hygroscopic amorphouscoating and may increase the amount of residual water vapor available tohydrolyze and/or plasticize the amorphous matrix, thereby reducing theglass transition temperature and increasing molecular mobility as wellas the rate of all chemical reactions in the solid. As the foregoingexample illustrates, various embodiments of the present inventionprovide an effective means for providing a dry environment. TABLE 1Space Analysis for Standard Packaging Conditions (without Desiccant)STANDARD PACKAGING CONDITIONS RESIDUAL GAS ANALYSIS 1 2 3 Mean s.d. CVPOUCH PRESSURE torr 684 586 749 673 82 12.2% NITROGEN ppm 987,275987,352 984,972 986,533 1,352  0.1% OXYGEN ppm 3,259 3,289 2,434 2,994485 16.2% ARGON ppm 169 158 133 153 18 12.0% CO2 ppm 119 124 134 126 8 6.1% MOISTURE ppm 9,052 8,960 12,223 10,078 1,858 18.4% HYDROGEN ppm126 116 104 115 11  9.6% HELIUM ppm ND ND ND ND ND ND FLUOROCARBONS ppmND ND ND ND ND ND CALCULATED RH at 22° C.  31% 26%  45%  34% 10%   29%CALCULATED RH at 50° C. 100% 98% 100% 100%*RH calculated at a temperature of 22° C., samples analyzed at ambienttemperature.

TABLE 2 Effect of Assembly Components on Pouch Head Space RESIDUAL GASANALYSIS +pre-dried ring Array Only POUCH PRESSURE torr 651 730 NITROGENppm 998,805 997,396 OXYGEN ppm 514 246 ARGON ppm 53 44 CO2 ppm 60 37MOISTURE* ppm 514 2,212 HYDROGEN ppm 54 65 HELIUM ppm ND NDFLUOROCARBONS ppm ND ND *CALCULATED RH 22° C. 2.2% 9.0%

TABLE 3 Effect of Added Desiccant on Pouch Head Space +Desiccant (2 ×125 mg +Desiccant RESIDUAL GAS ANALYSIS labels) (3.5 g sachet) POUCHPRESSURE torr 736 624 NITROGEN ppm 977,856 982,666 OXYGEN ppm 7,46315,335 ARGON ppm 367 617 CO2 ppm 207 1,237 MOISTURE* ppm 14,012 41HYDROGEN ppm 95 47 HELIUM ppm ND ND FLUOROCARBONS ppm ND ND *CALCULATEDRH 22° C. 51% 0.12%

TABLE 4 Internal Head Space Analysis on Macroflux ® PTH Systems inCurrent Packaging Configuration (Coated array, Tyvek pouch, 3 Å, 3.5 gDesiccant Sachet, Foil Pouch, N₂ Purge) RESIDUAL GAS ANALYSIS 1 2 3 4 56 7 8 9 10 11 12 POUCH torr 190 180 190 187 190 190 191 191 191 187 187191 PRESSURE NITROGEN % 98.1 98.1 98.9 98.9 98.4 98.4 97.3 97.3 97.997.9 98.2 98.2 OXYGEN % 1.79 1.77 1.00 1.00 1.54 1.54 2.52 2.52 2.042.02 1.67 1.68 ARGON ppm 784 785 431 435 688 666 1,112 1,102 888 899 739741 CO2 ppm <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100MOISTURE* ppm <100 <100 <100 <100 162 258 <100 <100 <100 <100 352 275HYDROGEN ppm <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100*CALCULATED 22° C. <1% <1% <1% <1% <1% <1% <1% <1% <1% <1% <2% <1% RH

Example 2

This example applies a delivery device having stratum corneum-piercingmicroprojections coated with a formulation of hBNP(1-32) (human b-typenatriuretic peptide). Table 5 outlines the experimental stability studydesign for this example.The effect of reducing moisture and oxygen wasassessed within the sealed pouch by addition of desiccant, oxygenabsorbers or pre-drying the system components prior to fmal packaging.An additional forced degradation parameter is terminal sterilizationwhich was assessed to generate accelerated stability data. Systems werepackaged into each of the five packaging configurations outlined inTable 6 and subjected to e-beam treatment at a dose of 15 or 21 kGy andambient temperature. Long-term stability samples for Groups A and B werestored at 2-8° C. or 25° C. and ambient humidity for up to 12 monthswith 5 time points. All other groups were stored at 25° C. and ambienthumidity for one month. One sample from each group was submitted forhead space analysis. All other samples were analyze by RP-HPLC forchemical stability by RP-HPLC and were compared to similarly packagednon-irradiated shipping controls.

The head space data is summarized in Table 7 along with the details ofthe packaging and treatment conditions. The moisture vaporconcentrations were converted to % RH assuming 760 torr total pouchpressure and 5 or 25° C. The non-irradiated shipping control (sample #1)was measured in triplicate to determine an estimation of therepeatability of the measurement system. For all gases detected at >500ppm the repeatability was ˜1% and for gases detected at levels <500 ppmthe repeatability was ˜10%.

The level of moisture for all samples containing desiccant are in-linewith prior head space analysis from similarly packaged systems withoutTyvek (˜100 ppm) indicating that the addition of the Tyvek pouch doesnot introduce a significant amount of moisture into the pouch headspace. The level of oxygen, however is slightly higher than previouslyobserved for a comparable system 0.2-0.3% vs 0.5% for this study priorto irradiation treatment. Following irradiation of the control system(packaging configuration A), the level of moisture appears unchanged,while the oxygen drops by a factor of ten and the hydrogen increases bya factor of 25. Interaction of the e-beam radiation with the polymericspecies in the pouch (Tyvek and/or polycarbonate ring) to generatefree-radicals and hydrogen as a by-product can reduce oxygen levels. Thefree-radicals that are generated can then react with oxygen in the headspace to form reactive peroxy radicals resulting in oxidativedegradation of the peptide

The two samples packaged under vacuum (sample #2 and #3) were lesseffective in purging the pouch of oxygen or, to a lesser extent,moisture. These samples had roughly 30 fold more oxygen in the headspace following irradiation, which may have been even higher prior toirradiation. Samples 5, 6 and 7 had no detectable levels of oxygen mostlikely due to the vacuum pre-treatment of the desiccant followed bynitrogen backfill for Sample 5, the and the lack of retainer rings forSamples 6 and 7, which are known to be a large source of oxygen. Thesomewhat higher moisture level (11% RH is still considered very dry)detected in Sample 6 is due to the absence of desiccant. Sample #7(untreated desiccant and no retainer ring) had no detectable level ofoxygen. In sample 7, oxygen released by the desiccant can be, consumedby free-radicals following e-beam treatment, notwithstanding the absenceof a retainer ring.

Three samples from each packaging condition were submitted for RP-HPLCpurity analysis immediately following e-beam treatment at T=0. Thesamples were averaged together and are summarized in FIG. 8 below andthe results are compared to the head space moisture and oxygen in Table8. The degradation profile for each condition is summarized in FIG. 9.

Purity decreases 2-3% following e-beam treatment at 15 or 21 kGy, thisdecrease is further exacerbated by increased levels of moisture in thepouch and to a lesser extent, high levels of oxygen. In addition tooxidation as the major degradation species, the acetate/citratemodifications observed at high retention times on the HPLC were alsoequally prevalent for this study. The modifications are a collection ofseveral smaller peaks that are combined to give the reported value.These levels of acetate modifications have not been observed previouslyunder similar e-beam treatment conditions (FIG. 10). One differencebetween the samples in present study is the Tyvek inner pouches (exceptsample #7) while samples in previous studies were packaged withoutTyvek. However, it is unlikely that Tyvek itself causes any additionalacetate/citrate modifications as sample #7, packaged without Tyvekcontains approximately the same level of degradation in this region(2.0%) as other samples packaged with Tyvek (average=2.2%).

Samples from each pouching/irradiation condition were stored for 4 weeksat 2-8 or 25° C. n=3 samples for each condition were analyzed by RP-HPLCand an average peak area percentage is presented in the figures below.FIG. 11 compares the T=0 total peak area hBNP purity values with the 1Mstability data packaged under nitrogen purge or vacuum.

The non-irradiated controls were also stored under acceleratedtemperature conditions of 40° C. for comparison. The data indicate thatin addition to damaging the peptide at T=0 during the irradiationprocess, e-beam treatment also increase the rate of degradation to wellabove the accelerated storage temperature of 40° C. for thenon-irradiated controls (light blue bar in FIG. 11). The degradationprofiles for the one month samples is presented in FIG. 12 and iscompared to the T=0 data for each condition.

The major degradation species for each condition is oxidation andacetate modification. While the hBNP(3-32) fragment was present for allconditions, it makes up on ˜0.5-1.0% of the total degradation peak area.Packages sealed without nitrogen purge under vacuum appear to be muchmore susceptible to oxidation, while pouches sealed under nitrogen showless oxidation. The acetate modifications do not appear to besignificantly affected by the packaging conditions. Table 9 summunarizesthe 1M stability data at 25C for all packaging configurations tested.

Compared to the control samples (Sample #1), packaged hBNP coatedsystems in the Tyvek inner pouch with desiccant and nitrogen purgeirradiated with 15 kGy e-beam resulted in <3% of hBNP degradation(Sample #2). More degradation was observed in Samples #3 & 4, which werepackaged under vacuum without nitrogen purge. At 21 kGy, the % purity isalready below the 90% specification. This data indicates that the highoxygen content affects peptide stability upon irradiation. Samples #5-7show better stability at T=0, particularly for Samples 5 & 7, which bothdemonstrate extremely low levels of moisture and oxygen.

Following storage for 1M at 2-8C, systems in packaging configuration #2irradiated at 15 kGy retained 93.1% total purity with mild totaloxidation at 1.4%, which is only 0.8% above non-irradiated controlsstored under the same conditions (Sample #1). The acetate/citratemodifications however increased to 2.2% as a result of the e-beamtreatment. For the systems packaged under vacuum rather than nitrogenpurge (Samples #3 & 4), both oxidation and acetate/citrate modificationsincreased above the non-irradiated control values. Systems packagedunder vacuum and irradiated at 15 or 21 kGy fell below the 90% totalpurity threshold following 1M storage at 2-8C mostly due to the BNPoxidation as a result of the high oxygen head space content.

Systems stored at 25C exhibited some increase in both oxidation andacetate/citrate modifications due to the elevated storage condition ascompared to similarly packaged/irradiated systems stored at 2-8C. Theadditional packaging measures of vacuum treating the desiccant, storagewithout assembly components and storage without Tyvek pouch mitigatedthe oxidative degradation process but had little affect on the amount ofacetate/citrate modification formed during storage at 25C even forsystems maintained in very low humidity and low head space oxygenenvironment (Sample #7).

Effect of Added Oxygen Absorbers

Additional hBNP (1-32) coated delivery systems were subjected to e-beamtreatment in various pouching configurations with and without theaddition of an oxygen absorber specifically designed to remove oxygenfrom the head space of the sealed foil pouch. Table 11 outlines thedifferent packaging configuration for this evaluation.

Systems from each condition were submitted for RP-HPLC purity analysisand head space following e-beam treatment (Table 12). All systems thatcontained an oxygen absorber (O2 scrubber) had very high humidity. O2scrubbers are iron based and can require higher humidity to catalyze theremoval of head space O2. For this reason, many O2 scrubbermanufacturers include moisture releasing salts with the O2 absorber tomaintain a relatively high humidity to allow the iron catalyst tofunction as intended and remove head space O2. All systems containingthe scrubbers achieved lower levels of oxygen compared to controlsystems.

Following e-beam treatment, samples from each group were analyzed byRP-HPLC purity assay. The results for total hBNP purity are presented inFIG. 13 and the degradation profile for the major degradation productsare presented in FIG. 14. The head space analysis is compared againstthe degradation profile in Table 13. Very low head space moisture (Run#6) or very low head space oxygen (Run #5) prevents degradation byoxidation or acetate modification during e-beam treatment. In addition,the presence of high levels of head space oxygen are not necessarilydamaging to the peptide unless there is sufficient level of moisturealso in the head space to facilitate the degradation mechanism. Run #7has very high levels of head space oxygen, however, the extremely dryenvironment (4% RH) prevents oxidation and or acetate modification ofthe peptide during e-beam treatment. Run #1 however, with a high levelof head space oxygen and a moderate level of moisture (41% RH) shows adramatic increase in the total oxidation (to 17%) with the level ofacetate modifications remaining at lower levels.

Run #4 and Run #9 samples were sealed without desiccant or O2 scrubber,Run #4 with nitrogen purge first, vacuum second and Run #9 with vacuumfirst then nitrogen. Both conditions performed equivalently with a totalpurity of ˜89% following e-beam treatment. For Run #8 and Run #10,samples were sealed with both 4A molecular sieve desiccant and O2scrubber. In Run #8, the pouches were first purged with nitrogen andthen vacuum, while pouches in Run #10 were first subjected to vacuum andthen purged with nitrogen. Again both sets of samples performedequivalently irrespective of the order of nitrogen purge or vacuum—totalpurity ˜92%.

Fitting the T=0 data from Terminal Sterilization Study #7 and Study #8into a linear least squares model indicated that hBNP oxidation wassensitive to the level of oxygen (P=0.0047) in the pouch but was notsignificantly affected by the level of moisture (P=0.9963). In contrast,hBNP degradation by the acetate modification pathways was more affectedby moisture (P=0.0002) and not head space oxygen (P=0.7260). Overall,the total BNP purity was affected significantly by moisture (P=0.0004)and less significantly by oxygen (P=0.0623).

Following one month storage at 25C, samples were analyzed by HPLC. Allresults reported are averages for n=3 systems packaged under similarconditions. FIG. 15 includes a comparison of the total HBNP purity asdetermined by RP-HPLC. Data from Run #6 and #7 were generated forTerminal Sterilization Study #7 as sample #2 and #4 respectively. Asanticipated from the T=0 stability data, Run #1 through #3 continued toperform poorly with most degradation due to the formation of acetatemodifications (FIG. 16) most likely due to the high relatively humidityinside the pouch. Table 14 summarizes the head space analysis and theT=1M 25C stability data. For packaging conditions that did not reducethe head space oxygen levels (Run # 1, 6 and 7), BNP oxidation continuedto increase above levels detected at T=0, doubling for these threeconditions following one month storage at 25C. All other packagingconditions, which maintained a low level of head space oxygen (<200 ppm)(Run #2-5 and 8-10), resulted in little to no detectable increase in BNPoxidation following on month at 25C. Therefore while not evident at T=0(e.g. Run #6 and 7), high levels of head space oxygen will lead to anincreased rate of BNP oxidation during storage. Therefore, maintaininglow levels of oxygen in the pouch is beneficial.

With the exception of Run #1 packaging configurations which containedhigh levels of head space moisture contained high levels of acetatemodifications. Samples packaged under the negative control conditionswere severely degraded after one month storage at 25C, with a totalpurity of only 42%. Acetate modifications into other degradation speciescan result from advanced degradation/further decomposition.

The above data confirm that the condition of the pouch environment, interms of moisture and oxygen is an important contributor to BNPdegradation following e-beam treatment. Diagrams I and II attempt tosummarize the cause and effect relationship illustrated by theexperiments. If oxygen and moisture levels inside the pouch are high(>60% RH and 1000ppm O2), BNP will degrade readily to form oxidized BNPand acetate modifications during e-beam treatment. Minimizing bothmoisture and oxygen in the pouch reduces BNP degradation by bothmechanisms. However, both mechanisms are more sensitive to moisturelevels, which should be rigorously maintained at low levels to preservepeptide stability. TABLE 5 Packaging Conditions Matrix IrradiationStorage Conditions Group Packaging Conditions Conditions T = 0 TempTotal 1 M 3 M 6 M 9/12 M A Sealed foil pouch with None (shipping ✓ −20°C. ✓ ✓ — — — Tyvek inner pouch, controls) 2-8° C. ✓ ✓ ✓ ✓ ✓ NitrogenPurge  25° C. ✓ ✓ ✓ ✓ ✓  40° C. ✓ ✓ — — — 15 kGy/ambient ✓ −20° C. ✓ ✓ —— — temperature 2-8° C. ✓ ✓ ✓ ✓ ✓  25° C. ✓ ✓ ✓ ✓ ✓ 21 kGy/ambient ✓−20° C. ✓ ✓ — — — temperature 2-8° C. ✓ ✓ ✓ ✓ ✓  25° C. ✓ ✓ ✓ ✓ ✓ B Foiland Tyvek, Sealed 15 kGy/ambient ✓ 2-8° C. ✓ ✓ ✓ ✓ ✓ under vacuumwithout temperature  25° C. ✓ ✓ ✓ ✓ ✓ 21 kGy/ambient ✓ 2-8° C. ✓ ✓ ✓ ✓ ✓temperature  25° C ✓ ✓ ✓ ✓ ✓ C Vac. Desiccant 21 kGy/ambient ✓  25° C. ✓✓ — — — D Array Only (w/o desicc.) temperature ✓ ✓ ✓ — — — E Array andTyvek Only (no ✓ ✓ ✓ — — —

TABLE 6 Stability Protocol Vacuum ‘+’ = Nitrogen Purge/ Assembly Dryingof Group Tyvek pouch ‘−’ = Vacuum Components. Desiccant Desiccant FoilPouch A + + + − + + B + − + − + + C + + + + + + D − + − − − + E + + −− + +

TABLE 7 Summary of Head Space Analysis Sample Number 1 1(repeat)1(repeal) 2 3 4 5 6 7 NITROGEN ppm 994062 994254 994242 995935 853524828976 995220 997884 997064 OXYGEN ppm 5178 5039 5088 753 111750 148190ND ND ND ARGON ppm 402 393 396 458 9389 9199 425 106 190 CO2 ppm 187 172155 205 364 223 144 652 175 MOISTURE ppm 49 29 18 104 226 234 31 745 16HYDROGEN ppm 122 113 101 2545 24747 13178 4180 613 2555 R.H. (@25 C.,760 torr) % 0 0 0 0 1 1 0 3 0 R.H. (@5 C., 760 torr) % 1 1 0 2 4 4 1 110 Packaging Type A A A A B B C D E E-beam Treatement NA NA NA 15 kGy 15kGy 21 kGY 21 kGy 21 kGy 21 kGy Packaged with Desiccant + + + + + + +− + + = Nitrogen/− = Vacuum + + + + − − + + + Vac/N2 treated desiccant −− − − − − + − − Inner Tyvek Pouch + + + + + + + − + AssemblyComponents + + + + + + + − −

TABLE 8 Data Summary Head Space Head Space E-Beam hBNP TotalAcetate/Citrate Sample N₂ O₂ Moisture Oxygen Conditions Purity Oxidationmodification, # Purge Vacuum Desiccant Scrubber Comments (% RH) (ppm)(room temp) (%) (%) RRT >1.33 (%) 1 + − + − shipping control 0 5102 None96.4 0.8 0.1 2 + − + − 2 753 15 kGy 93.7 1.3 1.7 3 − + + − 4 111750 15kGy 91.2 2.9 22 4 − + + − 4 148190 21 kGy 89.6 3.1 3.2 5 + − + − Treateddesiccant 1 0 21 kGy 92.8 1.7 2.1 6 + − − − No assembly 11 0 21 kGy 91.32.2 1.8 components or desiccant 7 + − + − No Tyvek pouch 0 0 21 kGy 93.01.3 2.0 or assembly components

TABLE 9 One Month 25 C. Stability Data Summary. Head Head Space SpaceE-Beam 1 M, 25 C. 1 M 25 C., 1 M, 25 C. Sample N₂ O₂ Moisture OxygenConditions hBNP Total Acetate # Purge Vacuum Desiccant Scrubber Comments(% RH) (ppm) (room temp) Purity (%) Oxidation (%) modification 1 + − + −shipping control 1 5102 None 96.6 0.6 0.2 2 + − + − 2 753 15 kGy 91.52.3 2.6 3 − + + − 4 111750 15 kGy 85.7 6.8 3.0 4 − + + − 4 148190 21 kGy63.6 7.5 3.6 5 + − + − Treated desiccant 1 0 21 kGy 90.5 2.4 3.2 6 + − −− No assembly 11 0 21 kGy 91.4 1.8 2.9 components or desiccant 7 + − + −No Tyvek pouch 0 0 21 kGy 91.6 1.3 3.2 or assembly components

TABLE 10 One Month 2-8 C. Stability Data Summary. Head Space Head SpaceE-Beam 1 M, 2-8 C. 1 M 2-8 C., 1 M, 2-8 C. Sample N₂ O₂ Moisture OxygenConditions hBNP Total Acetate # Purge Vacuum Desiccant Scrubber Comments(% RH) (ppm) (room temp) Purity (%) Oxidation (%) modification 1 + − + −shipping 1 5102 None 96.9 0.6 0.0 control 2 + − + − 2 753 15 kGy 93.11.4 2.2 3 − + + − 4 111750 15 kGy 88.5 4.7 2.3 4 − + + − 4 148190 21 kGy87.0 4.7 3.7

TABLE 11 Packaging Study Design O2 Run Nitrogen Vacuum DesiccantScrubber Comments 1 − − − − N/A 2 + − − + N/A 3 − + − + N/A 4 + + − −Nitrogen 1^(st), Vacuum 2^(nd) 5 − − + + N/A 6 + − + − Previous study 7− + + − Previous study 8 + + + + Nitrogen 1^(st), Vacuum 2^(nd) 9 + + −− Vacuum 1^(st), Nitrogen 2^(nd) 10 + + + + Vacuum 1^(st), Nitrogen2^(nd)

TABLE 12 Head Space Analysis SAMPLE ID Run #1 Run #2 Run #3 Run #4 Run#5 Run #6 Run #7 Run #8 Run #9 Run #10 NITROGEN ppm 787951 973502 904959923021 969568 995935 828976 957206 994988 982434 OXYGEN ppm 187385 170189 172 ND 753 148190 130 149 132 ARGON ppm 9478 615 10725 3824 10784458 9199 2603 223 291 CO2 ppm 5420 151 158 14243 159 205 223 613 1344609 MOISTURE ppm 3336 21906 25126 2881 11509 104 234 12732 1318 13026HYDROGEN ppm 6430 3656 58843 55859 7980 2545 13178 26716 1978 3508 R.H.% 11 58 78 9 36 0 1 40 5 37 (@25 C., 760 torr) R.H. (@5 C., % 41 100 10036 100 2 4 100 15 100 760 torr) Packaging Conditions Irradiation 21kGy/RT 21 kGy/ 21 kGy/ 21 kGy/ 21 kGy/ 21 kGy/ 21 kGy/RT 21 kGy/RT 21kGy/RT 21 kGy/RT RT RT RT RT RT N2 Purge − + − + − + − + + + Vacuum −− + + − − + + + + Desiccant − − − − + + + + − + O2 Scrubber − + + − + −− + − + Comments Negative Nitrogen From TS From TS Nitrogen VacuumVacuum Control first #7 #7 first first first vacuum vacuum nitrogennitrogen second second second second

TABLE 13 Comparison between head space and T = 0 stability data.Acetate/Citrate Head Space Head Space E-Beam hBNP Total modification, N₂O₂ Moisture Oxygen Conditions Purity Oxidation RRT >1.33 Sample # PurgeVacuum Desiccant Scrubber Comments (% RH) (ppm) (RT) (%) (%) (%) Run #1− − − − Negative Conrtol 41 187385 21 kGy 75.0 17.3 2.0 Run #2 + − − +100 170 21 kGy 59.3 2.4 35.6 Run #3 − + − + 100 189 21 kGy 63.1 0.7 33.8Run #4 + + − − Nitrogen first 36 172 21 kGy 89.5 4.8 2.0 vacuum secondRun #5 − − + + 100 ND 21 kGy 92.4 1.2 2.5 Run #6 + − + − From TS #7 2753 15 kGy 93.7 1.3 1.7 Run #7 − + + − From TS #7 4 148190 21 kGy 89.63.1 3.2 Run #8 + + + + Nitrogen first 100 130 21 kGy 91.7 1.0 3.6 vacuumsecond Run #9 + + − − Vacuum first 12 149 21 kGy 88.6 4.5 2.7 Nitrogensecond Run #10 + + + + Vacuum first 100 132 21 kGy 92.3 1.1 2.8 Nitrogensecond

TABLE 14 Comparison between head space data and T = 1M, 25 C. stabilitydata. Head Head 1M Space Space E-Beam 1M, 25 C., Total 1M, 25 C. O₂Moisture Oxygen Conditions 25 C. hBNP Oxidation Acetate Sample # N₂Purge Vacuum Desiccant Scrubber Comments (% RH) (ppm) (RT) Purity (%)(%) modification Run #1 − − − − Negative 41 187385 21 kGy 42.18 37.102.59 Conrtol Run #2 + − − + 100 170 21 kGy 34.06 0.51 52.93 Run #3 − +− + 100 189 21 kGy 30.71 0.46 39.67 Run #4 + + − − Nitrogen first 36 17221 kGy 88.47 3.45 3.48 vacuum second Run #5 − − + + 100 ND 21 kGy 90.541.27 4.05 Run #6 + − + − From TS #7 2 753 15 kGy 91.49 2.31 2.56 Run #7− + + − From TS #7 4 148190 21 kGy 83.63 7.53 3.64 Run #8 + + + +Nitrogen first 100 130 21 kGy 90.15 1.38 4.48 vacuum second Run #9 + + −− Vacuum first 12 149 21 kGy 88.76 3.65 3.67 Nitrogen second Run#10 + + + + Vacuum first 100 132 21 kGy 87.79 1.14 7.22 Nitrogen second

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1. A method for manufacturing a transdermal delivery device; wherein themethod comprises the following steps: (i) providing a microprojectionmember having a plurality of microprojections; (ii) providing abiocompatible coating formulation comprising a biologically activeagent, (iii) coating the microprojection member with the biocompatiblecoating formulation to form said transdermal delivery device; and (iv)packaging said transdermal delivery device under dry inert atmosphericconditions and/or a partial vacuum.
 2. The method of claim 1, whereinpackaging the transdermal delivery device comprises providing adesiccant.
 3. The method of claim 2, wherein the desiccant comprisescalcium oxide, clay desiccant, calcium sulfate, silica gel or a mixturethereof.
 4. The method of claim 1, wherein the dry inert atmospherecomprises nitrogen.
 5. The method of claim 1 wherein the dry inertatmosphere comprises nitrogen, argon, helium, neon, krypton, carbondioxide, or a mixture thereof
 6. The method of claim 1, wherein the dryinert atmosphere has essentially zero water content.
 7. The method ofclaim 1, further comprising providing a purge device to reduce moistureor oxygen content during packaging.
 8. The method of claim 1, whereinpackaging said transdermal delivery device is carried out under partialvacuum.
 9. The method of claim 8, wherein partial vacuum is from about0.01 to about 0.3 atmospheres.
 10. The method of claim 1, whereinpackaging comprises providing a foil-lined chamber.
 11. The method ofclaim 10, wherein said transdermal delivery device is enclosed in saidfoil-lined chamber.
 12. The method of claim 10, wherein the chambercomprises a foil lid, and a desiccant and/or oxygen absorber is attachedto the foil lid wherein said chamber is sealed by the foil lid.
 13. Themethod of claim 12, wherein and the chamber is purged with dry nitrogenprior to sealing the chamber.
 14. The method of claim 1, wherein thebiologically active agent comprises HPTH.
 15. The method of claim 1,wherein the biologically active agent comprises hPTH(1-34) and analogsthereof.
 16. The method of claim 15, wherein packaging comprises heatsealing said transdermal delivery device in a foil pouch purged withnitrogen gas.
 17. The method of claim 16, wherein said transdermaldelivery device comprises a ring and said method comprises pre-dryingthe ring to effectively remove desorbed oxygen and/or moisture from thering prior to pouching.
 18. The method of claim 15, wherein saidpackaging comprises adding a desiccant to the pouch.
 19. The method ofclaim 18, wherein the desiccant comprises a 4 Å molecular sievedesiccant.
 20. The method of claim 19, wherein said pouch is a foilpouch, said desiccant comprises 3.5 g Minipax molecular sieves packagedin Tyvek sachets and 125 mg Desimax adhesive labels affixed to-insidewalls of the foil pouch.
 21. The method of claim 1, wherein thebiologically active agent comprises hBNP (human b-type natriureticpeptide).
 22. The method of claim 1, wherein the biologically activeagent comprises hBNP(1-32).
 23. The method of claim 22, whereinpackaging comprises heat sealing said transdermal delivery device in afoil pouch purged with nitrogen gas.
 24. The method of claim 23, whereinsaid transdermal delivery device comprises a ring and said methodcomprises pre-drying the ring to effectively remove desorbed oxygenand/or moisture from the ring prior to pouching.
 25. The method of claim22, wherein said packaging comprises adding a desiccant to the pouch.26. The method of claim 25, wherein the desiccant comprises a 4 Åmolecular sieve desiccant.
 27. The method of claim 26, wherein saidpouch is a foil pouch, said desiccant comprises 3.5 g Minipax molecularsieves packaged in Tyvek sachets and 125 mg Desimax adhesive labelsaffixed to the inside walls of the foil pouch.
 28. The method of claim1, wherein said packaging comprises sterilizing said transdermaldelivery device.
 29. The method of claim 28; wherein said sterilizingcomprises subjecting said transdermal delivery device to e-beamtreatment.
 30. The method of claim 1, wherein the biologically activeagent is selected from anti-infectives, analgesics, analgesiccombinations; anesthetics; anorexics; antiarthritics; antiasthmaticagents, anticonvulsants; antidepressants; antidiabetic agents;antidiarrheals; antihistamines; anti-inflammatory agents; antimigrainepreparations; antimotion sickness preparations; antinauseants;antineoplastics ; antiparkinsonism drugs; antipruritics; antipsychotics;antipyretics; antispasmodics; anticholinergics; sympathomimetrics;xanthine derivatives; cardiovascular preparations; beta blockers;beta-agonists; antiarrythmics; antihypertensives; ACE inhibitors;diuretics; vasodilators, central nervous device stimulants; cough andcold preparations; decongestants; diagnostics; hormones; hypnotics;immunosuppressants; muscle relaxants; parasympatholytics;parasympathomimetrics; prostaglandins; proteins; peptides;psychostimulants; sedatives; tranquilizers; vasoconstrictors,anti-healing agents and pathway patency modulators.
 31. The method ofclaim 1, wherein the biologically active agent is selected fromantibiotics; antiviral agents; fentanyl, sufentanil, remifentanil,buprenorphine; terbutaline; scopolamine; ondansetron; nifedipine;dobutamine; ritodrine; atenolol; ranitidine; and parathyroid hormone;growth hormone release hormone (GHRH), growth hormone release factor(GHRF), insulin, insultropin, calcitonin, octreotide, endorphin, TRN,NT-36 (chemical name: N-[[(s)-4-oxo-2-azetidinyl]carbonyl]-L-histidyl-L-prolinamide), liprecin, pituitary hormones (e.g.,HGH, HMG, desmopressin acetate, etc), follicle luteoids, aANF, growthfactors such as growth factor releasing factor (GFRF), bMSH, GH,somatostatin, bradykinin, somatotropin, platelet-derived growth factorreleasing factor, asparaginase, bleomycin sulfate, chymopapain,cholecystokinin, chorionic gonadotropin, erythropoietin, epoprostenol(platelet aggregation inhibitor), glucagon, HCG, hirulog, hyaluronidase,interferon alpha, interferon beta, interferon gamma, interleukins,interleukin-10 (IL-10), erythropoietin (EPO), granulocyte macrophagecolony stimulating factor (GM-CSF), granulocyte colony stimulatingfactor (G-CSF), glucagon, leutinizing hormone releasing hormone (LHRH),LHRH analogs; goserelin, leuprolide, buserelin, triptorelin,gonadorelin, napfarelin, menotropins (urofollitropin (FSH) and LH)),oxytocin, streptokinase, tissue plasminogen activator, urokinase,vasopressin, deamino [Va14, D-Arg8] arginine vasopressin, desmopressin,corticotropin (ACTH), ACTH analogs such as ACTH (1-24), ANP, ANPclearance inhibitors, angiotensin II antagonists, antidiuretic hormoneagonists, bradykinn antagonists, ceredase, CSI's, calcitonin generelated peptide (CGRP), enkephalins, FAB fragments, IgE peptidesuppressors, IGF-1, neurotrophic factors, colony stimulating factors,parathyroid hormone and agonists, parathyroid hormone antagonists,parathyroid hormone (PTH), PTH analogs such as PTH (1-34), prostaglandinantagonists, pentigetide, protein C, protein S, renin inhibitors,thymosin alpha-1, thrombolytics, TNF, vasopressin antagonists analogs,alpha-1 antitrypsin (recombinant), and TGF-beta.
 32. The method of claim1, wherein the biologically active agent comprise a vaccine or antigen.33. The method of claim 1, wherein the biologically active agent isselected from bacteria, protein-based vaccines, polysaccharide-basedvaccine, nucleic acid-based vaccines, antigens in the form of proteins,polysaccharide conjugates, oligosaccharides, and lipoproteins.
 34. Themethod of claim 1, wherein the biologically active agent is selectedfrom Bordetella pertussis (recombinant PT accince—acellular),Clostridium tetani (purified, recombinant), Corynebacterium diptheriae(purified, recombinant), Cytomegalovirus (glycoprotein subunit), Group Astreptococcus (glycoprotein subunit, glycoconjugate Group Apolysaccharide with tetanus toxoid, M protein/peptides linke to toxingsubunit carriers, M protein, multivalent type-specific epitopes,cysteine protease, C5a peptidase), Hepatitis B virus (recombinant PreS1, Pre-S2, S, recombinant core protein), Hepatitis C virus(recombinant—expressed surface proteins and epitopes), Humanpapillomavirus (Capsid protein, TA-GN recombinant protein L2 and E7[from HPV-6], MEDI-501 recombinant VLP L1 from HPV-11, Quadrivalentrecombinant BLP L1 [from HPV-6], HPV-11, HPV-16, and HPV-18, LAMP-E7[from HPV-16]), Legionella pneumophila (purified bacterial survaceprotein), Neisseria meningitides (glycoconjugate with tetanus toxoid),Pseudomonas aeruginosa (synthetic peptides), Rubella virus (syntheticpeptide), Streptococcus pneumoniae (glyconconjugate [1, 4, 5, 6B, 9N,14, 18C, 19V, 23F] conjugated to meningococcal B OMP, glycoconjugate [4,6B, 9V, 14, 18C, 19F, 23F] conjugated to CRM197, glycoconjugate [1, 4,5, 6B, 9V, 14, 18C, 19F, 23F] conjugated to CRM1970, Treponema pallidum(surface lipoproteins), Varicella zoster virus (subunit, glycoproteins),and Vibrio cholerae (conjugate lipopolysaccharide).
 35. The method ofclaim 1, wherein the biologically active agent is selected from weakenedor killed viruses; vaccines comprising nucleic acids include;single-stranded nucleic acids; double-stranded nucleic acids,supercoiled plasmid DNA; linear plasmid DNA; cosmids; bacterialartificial chromosomes (BACs); yeast artificial chromosomes (YACs);mammalian artificial chromosomes; RNA molecules, and mRNA.
 36. Themethod of claim 32, wherein the biologically active agent furthercomprises one or more augmenting adjuvants together with the vaccine orantigen.
 37. The method of claim 36, wherein the one or more augmentingadjuvants are selected from aluminum phosphate gel; aluminum hydroxide;algal glucan: b-glucan; cholera toxin B subunit; CRL1005: ABA blockpolymer with mean values of x=8 and y=205; gamma inulin: linear(unbranched) β-D(2→1) polyfructofuranoxyl-a-D-glucose; Gerbu adjuvant:N-acetylglucosamine-(b1-4)-N-acetylmuramyl-L-alanyl-D-glutamine (GMDP),dimethyl dioctadecylammonium chloride (DDA), zinc L-proline salt complex(Zn-Pro-8); Imiquimod(1-(2-methypropyl)-1H-imidazo[4,5-c]quinolin-4-amine; ImmTherÔ:N-acetylglucoaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-glyceroldipalmitate; MTP-PE liposomes: C59H108N6019PNa-3H2O (MTP); Murametide:Nac-Mur-L-Ala-D-Gln-OCH3; Pleuran: b-glucan; QS-21; S-28463: 4-amino-a,a-dimethyl-1H-imidazo[4,5-c]quinoline-1-ethanol; sclavo peptide:VQGEESNDK.HCl (IL-1b 163-171 peptide); and threonyl-MDP (TermurtideÔ):N-acetyl muramyl-L-threonyl-D-isoglutamine, and interleukine 18, IL-2IL-12, IL-15, Adjuvants also include DNA oligonucleotides, such as, forexample, CpG containing oligonucleotides. In addition, nucleic acidsequences encoding for immuno-regulatory lymphokines such as IL-18, IL-2IL-12, IL-15, IL-4, IL 10, gamma interferon, and NF kappa B regulatorysignaling proteins.
 38. The method of claim 1, wherein the biologicallyactive agent further comprises one or more counterions to furtherimprove the stability of the formulation.
 39. The method of claim 38,wherein the one ore more counterions are selected from acetate,propionate, butyrate, pentanoate, hexanoate, heptanoate, levulinate,chloride, bromide, citrate, succinate, maleate, glycolate, gluconate,glucuronate, 3-hydroxyisobutyrate, 2-hydroxyisobutyrate, lactate,malate, pyruvate, fumarate, tartarate, tartronate, nitrate, phosphate,benzene sulfonate, methane sulfonate, sulfate, sulfonate, sodium,potassium, calcium, magnesium, ammonium, monoethanolamine,diethanolamine, triethanolamine, tromethamine, lysine, histidine,arginine, morpholine, methylglucamine, and glucosamine.
 40. A method fordelivering stable biologically active agent formulations, wherein themethod comprises preparing a transdermal delivery device according toclaim 1 and applying said transdermal device to the skin of a subject.41. A transdermal delivery deviced prepared by the method of claim 1.42. A method for delivering biologically active agent formulationscomprising: (i) providing a microprojection member having a plurality ofmicroprojections, (ii) providing a formulation of biologically activeagent; (iii) forming a biocompatible coating formulation that includesthe formulation of biologically active agent, (iv) coating themicroprojection member with the biocompatible coating formulation toform a biocompatible coating; (v) packaging the biocompatible coatingunder dry inert atmospheric conditions and/or a partial vacuum; and (vi)applying the coated microprojection member to the skin of a subject. 43.A method for manufacturing a drug delivery device; wherein the methodcomprises the following steps: (i) providing a substrate; (ii) providinga biocompatible coating formulation comprising a biologically activeagent, (iii) coating the substrate with the biocompatible coatingformulation to form said drug delivery device; and (iv) packaging saiddrug delivery device under dry inert atmospheric conditions and/or apartial vacuum.